Document jnG8ra7ZKJLrjB7Zdg1a7vER

ARR26- 066& PFOA Genotoxicity - | 000865 MUTAGENICITY TEST ON CORNINGHazleton T6358 MEACSHUIRNIENSEG HCAHMRSOTMEORSOOVMAARLYA(BCEHROR)ACTEILOLNSS IN ~~~ FINALREPORT AUTHOR Hemalatha Murli, Ph.D. NNeewnmaen 5 PERFORMINGLABORATORY `Coming Hazleton Inc. (CHV) 9200 Leesburg Pike Vienna, Virginia 22182 LABOPRROJEACTITDENTOIFIRCATYION CHV Study No: 17388.0437 SUBMITTETRO 3M3Cemnter St.Paul,Minnesota 55144-1000 STCUODYMPLETIDOATNE April 25,1996 CHV Study No.: 17388-0-437 lof24 000866 `CORNING Hazleton QUALITY ASSURANCE STATEMENT Project Title: Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells Project No.: 20990 Assay No. 17388 Protocol No. 437 Editon No.: 15 `pQruoajleicttywAesrseurcaonncdeucitnesdpeacctcioorndsoifngtthoe tshteudSytaannddarrdevOipeewroaftitnhge PfrioncaledruerpeorstofotfthheeQaubaloivteyrAesfseurernacnecde Urengiutlaatnidonasc.coFridnidnigngos fthreomgetnheerailnsrpeeqcutiiroenmseanntdsoffintahlereappoprrtoprreivaietwesGwoeorde Lraebpoorrtaetdortyo mPraancatgiceement and 10 the study director on the following dates: InspectionDate Harvess0214/1996 Eindings Regoned 0214/1995 Auditor S. Ballenger Draft Report Review/042021/1996~~ 0412211996 Final Report Review/04125/1996 04251199 C. Orantes C.Orantes Pa LEGZ[TED Joss Quality Assurance Unit Date Released CHV Study No.: 17388-0-437 2 000867 CORNINGHazleton STUDY COMPLIANCE AND CERTIFICATION fTohreth sitnutdhye wFaosodcoanndduDcrteudgiAndmcionmipsltiraantcieowni(thFDtAhe)TGiotoled 2L1aobfortahteorUy.SP.raCcotidcee orfegFueldaetriaolnsasset `Raemgeunldamteinotnss.PaTrthe5r8e.wiesrsueendoDescigenmifbiecran2t2.dev1i97a8t,io(nesfffercotmivtehJeuanfeo2r0e.me1n9t7i9o)newditrhegaunlyataipopnlsicoarbtlhee Tsihgenerdawprdoattoacohlavtheatbeweonulredvaifefweectd tbhye tihneteSgtriutdyoy fDitrheectsotru.dywhoor tcheretiifniteesrptrheattattihoenoevfaltuhaetitoesnt orfesutlhtes. test anticle as study design presented herein represenatns and evaluation criteria. appropriate conclusion within the contextof the All test and control results in record has been reviewed by tthheisStreupdoyrtDiarreectsourp.porAltledrabwydaantae,xpdeorciummeennttaaltidoant.a record and this records, protocol faancidalitceosopyfofCotrhneinfignaHlazrelpeotrotngIennce.rfaotredatalseasrtesounletyoefatrhifsolsltouwdiyngwisllubbmeisasrichoinovfedthinetfhienasltorreapgoert to mthaeteSrpioanlssorre.taiAnfetderitnhtehoensetoyreaagre pfaecriiloidt,estohefSCpoomnsionrgmHaayzleetloenctItonc.hafvoertahneaafdodriteimoennatliopnereido.d of time. or sent to a storage facility designated by the Sponsor. Submitted by: Study Director Hemalatha Murl, Ph.D. DMeapmamratlmienatnoCfyGteongeetnietciacnsd Cellular Toxicology Study Completion Date CHV Study No.: 17388-0437 3 000868 CORNINGHazleton TAOFBCOL NTENE TS Page No ABSTRACT 6 10 SPONSOR . . 7 20 MATE(TERSTAIRTAICLLE) ..... 7 21 Clients Idenification 22 DateReceived 23 Physical Description 24 Genetics Assay No. 30 TYPEOFASSAY orrrasesesasen . 7 40 PROTOCOLNO................... ~ oessssasdd 50 STUDY DATES ereteeree sagan rane 7 5.1 Initiation Date 52 ExperimSetanrttDaatle. 53 `Experimental Termination Date 60 SUPERVISORY PERSONNEL ........................... 7 61 Study Director 62 Laboratory Supervisor 70 OBJECTIVE . . cn . cen? 80 RATIONALE .................... rereennersnnnenen 8 90 EXPERIMENTALDESIGN ......... Ce 8 100 MATEARNDIMEATHLODSS - 101 Indicator Cells 102 CellCulture Medium 103 Negative and Solvent Controls 104 Positive Control Agents. 105 Rangefinding Assays 106 Aberrations Assay Without Metabolic Activation cd CHV Study No.: 17388-0437 4 000869 `CORNING Hazleton 107 Aberrations Assay With Metabolic Activation 108 Harvest Procedure 109 Slide Preparation and Staining 10.10 Aberrations Analysis and Assay Evaluation 110 RESULTS onprine ce cn ny Solubility and Dose Determination 3 nz Rangefinding Assay Without Metabolic Activation ns Rangefinding Assay With Metabolic Activation 114 Chromosomal Abemations Assay Without Metabolic Activation ILS Chromosomal Aberrations Assay With Metabolic Activation 120 CONCLUSION eb 15 130 REFERENCES wperasnn rs pssesasan . 15 140 EXPERIMENTAL DATA TABLES ce . 1" 150 DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS . ERE. wvrsasss -2 CHV Study No.: 17388-0437 Ss 000870 CORNING Hazleton ABSTRACT The objective `abermations in of ths in vitro assay was to evaluate Chinese hamsterovary (CHO) cells the abilityof T6338 to induce chromosomal withand without metabolic activation. dFiosrsotlhveeddosine srearnigleefdinediionngizaesdsawyastewriatth aancdonwcietnhtoruattimoentoafbolSi0c1 amcgt/imvalt.ionC.onthceenttesrtatairtoincsloefwas0.167. 0.501. 1.67. 5.01. 16.7. 50.1. 167. 501. 1670.and 5010 ug/ml were testedin the dose rangefinding assays. All dosing was `Complete cytotoxicity was observed achieved usinag dosing volume of 1% (10.0 ulmi) in the cultures dosed with 501. 1670. and 5010 ug/ml in the fnoenwacmteitvaapthiaosneassswaeyreanadvawiiltabhle16i7n0thaendcuSlt0u1r0e ig/ml in the treated with assay with 167 g/ml metabolic activation. and a severe cell cycle Very delay was observed. No cell cycle delaywas observed activation assay. in the cultures analyzed in the metabolic iBnacsuebdatoend twhiethdat12a.5f.ro2m5.t0h,e5d0o.0s.e 7r5a.n0g,ef1i0n0d.i1n5g0.asasanyd,2r0e0pligc/atmelcuilntaur2e0so.0fhCoHurOabceerllrsatwieornse assay w1i5t0h0ouutg/mmeltawbiotlhica a2c0t.i0vahtoiounr.abReerprlaitciaotnescauslstauyrewsiwthermeettarbeaotleidcwaictthiva1t2i5o,n.250C,ul5t0u0r,es75t0re,at1e0d00w,itahnd 75.0, with 100, 125. 150, 250. and 500. 200 and ug/mi from the 750 ug/ml from assay without the assay with metabolic metabolic activanadtculituorens treated activation were analyzed for chromosomal aberrations. No significant observed at the concentrations analyzed. increase in cells with chromosomal aberrations was `The cells twesitthanaincdlew.iTt6h3o5u8t,mewtaasbocloincsiadcetirveadtinoeng.ative for inducing chromosomal aberrations in CHO. CHV Study No.: 17388-0437 6 000871 `CORNING Hazleton Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells With T6358 10 SPONSOR: 3 20 MATERIAL (TEST ARTICLE): 21 Client's Identification: T6358 22 Dae Received: January 16.1996 23 Physical Description: White powder 24 Genetics Assay No.: 17388 30 TYPEOF ASSAY: Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells 40 PROTOCOLNO.: 437, Edition 15 50 STUDY DATES: 5.0 Initiation Date: January 18. 1996 52 Experimental Start Date: January 30. 1996 $3 Experimental Termination Date: March 20, 1996 60 SUPERVISORY PERSONNEL: 6.1 Study Director: Hemalatha Murl, Ph.D. 62 Laboratory Supervisor: Carol S. Spicer, B.S. 70 OBJECTIVE: iTnhdeucoebjcehctriovmeoosfotmhaisl ianbevrirtartoiaosnssaiynwCahsinteoseevhaalmuastteerthoevaabriyli(tyCHofOt)hecetlelsst, awnititchlea,nTd6w3i5t8h.outto `metabolic activation. CHV Study No.: 17388-0-437 7 000872 CORNINGHazleton 80 RATIONALE: "wTihtehacsesalylioss diensdiugcenecdhtrooemsotasbolmisehbwrheaektsh.erCthheemitecsatlalnytiilneduorceidtslmeestiaobnoslmiateys rcesaunltinitneract obrreraekssultinicnhvriosmibalteindatmhaagtea.re Acibtheerrrreaptaiiaroerdenabsyctohnesceeqllueinncseoufcahfaiwlauyrae sortomibsetauknedsetiencrteapbalier processes 1962). such that breaks do not ejoin or rejoin in abnormal configurations (Evans, The assay is a chemical. designed to This design elixmaimtsinleoscsoelflsabienrtrhaentfircsetllmsitdousriisngaftehre initiationof treatment division process or with cceolnlvs.ermsoisotn diinvtiodciongmpclelelxs deexraimviatnievdes8d-u1r2ihnogusrusbasfeeqruetnrteacetlmlenctycalrees.in ItnhethfeircstasmeitoofsiCsH(OM, cells). assay Many test articles cause severe hasbeen designed to detect this delayofprogression through the cell delay and allow for slower growth of cycle. and damaged the cells by adjustments in the time between treatment and cell fixation. 90 EXPERIMENTAL DESIGN: tRheesuclhtrsofmroosmomthaelraabnegrerfaitnidoinnsgaassssaayyawnedrteo udseetdertmoidneettehremionpetitmhaeldtoismeeorafnhgaervtoesbte oufsetdhein `deoxspeodsucreelltsostohethtaetstparritmiacrlielwyomueltdapbheaasneacleylzlesdwfhoircchhwreormeosionmtahle faibrestramteitoansp.hasIen stihnece dreaonxgeyfuirniddiinnge a(sBsradyU.rdt)h.e cuTlhtiusreesnawbelreeditnhceubmaatjeodriftoyro2f5c-e2l6lshtoourpsrowgirtehss5-tbhrroomuog-h2'a-bout two cterlelatdmiveinstiosncsh,edtuhlues pfrorovtihdeirnagnageffuilndaisnsgeassssmaeyntiosfgcievleln cbyeclloew.kinetics. A summaryofthe --_-- -- Test = Testicle T WashPBlrdUdrd WaWasshh CoCloeleeemmiidd FiFxiaxtatiioonn -s9 0 S22 sa 287 77 B --_+--59 0 7 2 2 7 257 27 SInintghleecchurltoumroessowmearle aubseerdraftoirotnhseasnseagyast,ivreepcloinctartoel,cuslotluvreenstwceornetruosl,edanatdeaatcehadchosoefltewvoe.l. dacotsievsatoifont.he20pohsoiutirvheacrovnetsrotls.weIrnetchoenadbuecrtreadt.ionCsharsosmaoyswoimtahlanadbewrirtahtioountsmweetarbeolainca:lyzed cfornotmrotlhedocsuelst.ureAs struemamteadraytotfhtehfeoutrrehaitghmeesntt sdcohseedluelveelfsoranthdefcrhormoomnoesoofmtahleabpeosrirtaitvieo.ns assays is given below. CHV Study No.: 17388-0437 3 000873 ' CORNING Hazleton `SummaryofChromosomalAberationsAssasTsaumentScheduleinHous Ter TemAmce | Wah | Colemid Fiaion "so 1s is 0 -s0 28x 100 MATERIALS AND METHODS 101 Indicator Cells, pTehremCahniennetsceehlaminstearnodvwareyrceolrlisg(inCalHlOy-oWbBaiLn)edusfdomn tthheisaabsosraytowreyreoffDrr.o5am.. Wolff. UniversityofCalifomia. San Francisco. The cells have since been orFec1l2o1ne0d1t4o hmoaiunstawiinthkarymootdyapliccshtraboimliotsy.omTehinsumceblelrloifneh2a1s anaverage cycle time 102 Cell Culture Medium, psTeuhnpeipcClileHlmiOennatcneelddlsswewieptrteo1mg0ry%ocwiennailantbMaocpvpCironoyex'issmear5tuaemlcyu(lF3tB7uSr)eC..m1ein9daiLgnulmautwamhomisicpnhheew.raesaonfds1ho%ut $40, nai. 103 Negaive and Solvent Conrls ef10olrtlhheanntodensctaucalitnvucarleti,moesnditaussema.yds.eSionolenvgieaztneitdvcewoacntotenrrto.rlosalwstehwreeehrciuglchtueulortseusrceocsnocwenhnintcriahntcigoonnhteuasinesodalnvlenystt csaotlintvruaetssiow(ne1m%ri,xtih.i.en.csl1au0dmeeldams.descIrnitbheedaicntihveatnioonnaaaivya,tohne<nsesgyatsivbeutawnidthsohveen$t9 104 Posive Control Agents a(TchMteiMvCpaot)siioftnorithsveeenrciooensntaroMcliitavogeamnyitcosinwniCscer(hiCeswAeaSrn#ed5uc0sy0ce7l.oi7np,htoShseipgahmsaasma.yiLsdoewte(4rCeP2)m5iiHnt0ohGme1y9cmie0nt.Calic 6c0la5s5t1o9g-en2t.aStidgmoae,sLoott #re6q7uiFrOe1SmeSt)abdoolciscnaocttiavtatidoinr.etClyycbluotpmhoaspbheamciodem(eCArSt#o CHV Study No. 17388.0.437 5 000874 : `CORNING Hazleton activ intermediates by microsomal enzymes. In the rangefinding assay. two tchoenccehnrtroamtoisoonsmoalfaMbMerCrat(i0o.n2s5asasnadys0..5t0wog/cmoln)ceanntrdatCiPons(15o.0f MaMndC20(.00.p0g8/amnid. In a0.10beurragtio/nsmainnltdhCe)PCH(O10.ce0llasn.d O1n5.e0ofgt/hme)dwoseereleuvseelsd wtaosinadnuacleyzcehdroinmoeascohmoafl the aberration assays. Both MMC and CP were dissolved in water. 105 Rangefinding Assays: wIneatthmeseentasbsyayss.eetdhiengceallpsprwoexriemactueltluyr0e.3d foxr1a0pprpeorxi2m5atcemlyl24akhoiunrtsoprimoir otof complete McCoy's 5a culture medium. 105.1 Assay Without Metabolic Activation: 2`T-hdeeocxulytuurriedsiwneer(eBdrodsUerddw) iwahsthaeddteedstaatrtaicflienfaolrc2o.nc2enhtoruartsiownheofn 1S0-burMo.moaTnhde fcruelsthurceosmwpelreteewmasehdeidum23c.o3nthaoiunrisnlgatBerrdwUirtdh apthoafsipnhaaltecobnucfefnetrreadtisoalnionef c1u0ltuuMre,sawnedreCotlhceenmhiadrve(sftienadltcwooncheonutrrastliaotner0a.1ndpgdi/fmfle)rewnatisalaldydetda.ineTdhefor (thFePGan)altyescihsnioqfucel(lSceyecSleecdteiloanys uosninHgaravemsotdiafnidedSlfildueoPrreespcaernactei-opnluasn-dGiemsa Staining). 1052 Assay With Metabolic Activation: aItn =th3i7saCsisanyt,htehepCreHseOncceelolsfawearteleixvepros9edrteoactthieontemsitxrtiurlee(f9or 1t5woulhmoiu,rs (NMAolDePcu1l.a5rmTgox/imc,olaongdy.isIoncc.i,tiLcotac#i0d528.37)mwg/amsi)d.eriTvhedefSrOomfrtachteiolniver of m1a2l5e4 1S0priangduucee-Dtahwelmeiyxeradtsfwunhcitcihohnaodxbideaesne epnrezvyimoeusslwyheisctheadrewciatphaAbrloecolfor t`miemteawbaolsiuzisnegdcbheecmaicuaslesp1r0olmoonrgeedacetxipovsfuorremst.o tTheheS9twmoixhtouurreimnicugbhattiboen. `tTohxeicmteodtihuemcedlilds anontdhtaheveenFzBySmdeuarcitnigvtihyeoefxSp9osiusrleospterriapoiddtloyaavto=i3d7C. pporsosdiubcleedinbaycttihveaSti9oneonfzsyhmoerst bliyvbedinadnidnghitgohsleyrruemacptriovteeiinnst.ermediates CHV Study No.: 17388.0437 10 000875 CORNING Hazleton sfaleinre. thCeoemxpploestuereMpceCroiyo'dsth5e2 cmeeldsiwuemrecwoaasihneidngtwBicredUwridth(fbiunfaflered concentration incubated for 2150.5uhMo)urwsawsiatdhdCeodl(c0emthiedcul(ftiunraelscwohnciecnhtrwaetrieonth0e.1n ug/ml) asldiddeeds wfoerrethperaespatr2e.0dhaonudrst.ainTehdeacsulwtausredsewsecrriebetdhefnorhatrhveesntoenda.ctfiivxaetd.ioannd rangefinding assay. 1053 Assay Evaluation: mOneet.aphhuansdersefdrcoomnstehecuatsisvaey mweittahpohuatsaensdrwoitmhtmhetcabdoolsiec laecvteilvsatwiiotnh(103 nfurommbetrhoefscoellvlenctyccolnetsrtolhrroougmhtwhhiacchtitvhaticoelnlsashsaady)prwoegrreesassesedswsheidlfoe inthtehe presenceof BrdUrd. 106 Aberrations Assay Without Metabolic Activation: Cinutlotu1r0esmlweorfecoinmiptliaetteed bMycCsoeeyd'isngSaapmperdoixuimm.ateOlnye1.d2axy a1t0erceclullstupreer i75nactmi?ofnlatshke cheolulrss.weTrheeicnuclutbuarteesdwaetre=t3h7enC wwiatshhetdheweistthbaurftficelreeadtsaplriendeetaenrdmicnoemdpdloestees for 17.5 cMecllCs.oyT'wso52homuerdsiluatmerc,otnhteaienlilnsgw0e.r1ewhga/rmviesCtoeldacenmdidirwdarsiepdlsalcieddesbwaecrkeomnaodet.he Tchhreosmiodseosmawelraebetrhreantisotnasi.ned in % Giemsa solution for the analysis of 107 Aberrations Assay With Metaboli Activation: Cinutlotu1r0esmlwoerfecoinmiptliaetteed bMycCsoeeyd'isngSaapmperdoixuimm.ateOlnye1.d2axy a1f0erceclullstupreer i7n5itciamt?ionf,latshke ctuhletSur9esrewaecrtieoinnmciuxbtautreed iant M=c3C7oyC'fsorS2a mheoudrisiunm twhiethporuetseFnBcSe.oftAfhieerttehset2arthicoluerand ewxeproerseufreedpweiritohd,ctohmeplceeltles MwcerCeoyw'assSheadmtewdiicuemw.itThhbeufcfeelrlsedwsearleiniencaunbdattheedcfeolrlsthe pfresetsoefntt dhurciunlgttuhre pleartio2d.0uphotuorsthoeftiinmceuobfathiaorn.vesTthweimthet0a.p1hgas/emcelCloslweeermeitdhen harvested and prepared for cytogenetic analysis. CHV Study No: 17388-0437 n 000876 `CORNING Hazleton 10.8 Harvest Procedure: TPrhieosretoobtsheervhaatrvieosnts oifnctlhuedceudlatnuraesss.evsissumaelntoobsfertveatpieorncsoenfttcoxoincfiltuyenwceeroefmtahdee.cell p`mroensoelnacyeoerfmwiittohitnicth(elacrugleturroeunfldaeskds.celTlsh)eorcduletaurdescewlelsreflaolastoinegvailnutahteedmefdoirutmh.e The m1e9t6a1p)haansde wceelrles twreerateecdowliltehct0e.d0b7y5 aMmiKtoCt!ichysphoatkoen-iocffs(olTuetriaons.imTahiasndtrTeoaltmmaencth. thheelnpsfitoxesdwewlilthtahencaebllssolauntde mtehtuhsadnioslp:ergsleactihaelcahcreotimcosaocimdes(,3:1,Thviev)cuflitxuarteisvewearned were washed several times before air-dried slides were prepared. 10.9 Slide Preparation and Suining; sSlliiddeess fwreorme tphreepraarnegedfbiynddirnogpapsisnagysthweehraervdiefsfteerdenctiualllytsoiunicnreledeausnsisnlgidaesm.odTihfeied 2f1.luo1r9e7s8c)e.ncTeh-eplsulsi-deGsiewmesrae(sFtPaGi)netdeicnhnHioqeucehs(atft3e3r2P5e8rrsytaainn,deWxoplofsfe.d1t9o74ul:trGaovtioo,leett alibgehrtr.aatinodnsthaesnsasytawineerdewsittahinGediewmistha 5Az%urGeieBmsstaains.oluTthioensfloirdetshepraenpaalryesdisforfom the umistiontgiDcecpherxomomsooumnatlinagbemreradtiiuonms.. All slides were then air-dried and cover slipped 10.10 Aberrations Analysisand Assay Evaluation: c(Ceenltlsrowmeerreesseelqeucatledtofotrhgeomooddamlornpuhmobleorg2y1an=d2o(nrlayncgeell1s9w-i2t3h) the number of were analyzed. aOnndefhruonmdrtehde nceeglastfirveomaenadcsholrveepnlticcaotnetrcoulltcuurletautrefsouwrerdeosaenalleyvezlesdoffotrhettheesdtifafretriecnlte, ccpueleltlssurowesef.rceFhaornroamclooynsztoerdmoalfloorfabcbieharrsro,amtaoilolsnossmli(adElevsaanbesexr,crea1pt9tif6oo2tn;shSfeereompSoeoscnitteiioovnfet1ch5oe.n0t)pr.ooslAisttwiveleeraecstocn2ot5rdoeld `prmiiocrrotsocaonpaelysstiasg.e Cells with location. aberrations were recorded on the data sheets by the c`Thhreofmoolsloomwianlgafbaecrtroartsiownesrdeattaa:ken into account in the evaluationofthe: 1. The overall chromosomal aberration frequencies. CHV Study No.: 17388-0437 2 000877 CORNING Hazleton 112 Rangefinding Assay Without Metabolic Activation Fthleoactuilntgurdeesbdrosiesndo.wcietlhl m1o6n7o0laaynedrs$.01a0ndugn/omlv.isiUbnlheemailttohtiyccceellllmsowneorleayoebrs.erfvleoadtiinng debris. =159% reduction in the cell monolayer confluence. and no visible mitotic `cemlolnsolwaeyrere.obfsloeartviendg dinebtrhies.cu=l1tu5r%e trreedautcetdiwointihnSt0h1e cpegl/lmml.onoUlnahyeearltchoynfcleulelnce. and scuelvteurrerterdeuacttediownistihn 1t6h7e ng/ummlb.er o_fTovxiisciibtlyewmaitsotmiacnicfeellsstewderoenotbhseesrlvieddesipnrtehpeared wferroem etvhaslucualtteudreinbtyhethceulatvuarielsabtirletaytoefdownitlhy1869.7,me5t0ap.h1asaensd. 16C7elplgc/ymclle(Tkaibnleetic1s) Severe cell cycle delay was observed in the culture dosed with 167 ug/ml. Based o25n.t0h.e5s0e.0r,es7u5l.ts0.,a10200.h1o5u0r. haanrdve2s0t0wgas/smeleicnttehdefnoorntaecsttivnagtcioonncaebnetrrraattiioonnssoafss1a2y..5. 113 Rangefinding Assay With Metabolic Activation Floating debris, no cell monolayers, and no visible mitotic cells were observed in othbesecruvlteudreisn adonsyoedftwhithot1h6e7r0caulntdurSes0.10Cge/llmlc.yclNeokivniestuiacls swiegrnesoefvatlouxaitceidtyinwetrhee cultures treated with 50.1, 167. and 501 ug/ml (Table 1). No cell cycle delay was. oseblseecrtveeddfiontthesetciunlgtcuornecseanntarlaytzieodn.soBfa1s2e5d. o2n50t.he5s0e0.re7su5l0t.s.1a00200, haonudr 1h5a0rv0egst/wmasin the aberrations assay with metabolic activation. 114 Chromosomal Aberrations Assay Without Metabolic Activation Usenvheeraeltrhedyuccetlilonmsonionltahyeernsu,mb=e3r0of%vriesdiubclteiomnitionttihcecceelllls mweorneoloabyseerrvceondfilnuetnhcee, and culture treated with 200 ug/ml. Toxicity was manifested on th slides prepared offrommettahpehsaesecsu.ltuUrenshebayltthheypcreellsemnocneoloafymearns,y=in1t5e%rprhaesdeuccteilolsn ainndthsepacerlslemnounmoblearyser confluence, and reductions in the numberofvisible mitolic ces were observed in threeduccutlitournesitnrtehateednuwmitbher1s5o0fuvgi/smilb.le Smliitgohttilcy cuenlhle,alatnhdy=c1el5l %morneodluacyteirons,insitghhetcell Cmohnroolmaoyseormcaolnfalbueerrnacteiownesreweobrseeervvaelduaitnetdhefrcoulmtutrhee tcrulattuerdeswitrtehat1e0d0wpigt/hml7.5.0, c10h0r.om1o50s.omaanld 2ab0e0rruagt/iomnls(wTaabsloeb2s).ervNeod astigtnhieficcoanncteinntcrraetaisoensinancaellyszewdi.th CHV Study No.: 17388-0437 1 000878 CORNING Hazleton `The sensitivityof the cell culture for inductionof chromosomal aberrations is msihtoowmnycbiynthCe. itnhcerpeoassietdivferecqounetrnocly aogfenatb.errTahteiotnesstinantihcelceelislsceonxspiodseerdedtonegative for inducing chromosomal aberrations under nonactivation conditions. 115 Chromosomal Aberrations Assay With Metabolic Activation dNoosecedllwimtohno1l5a0y0eurgs/maln.d nDoeavidsicbellel mmiotnootliacyceerlsls, wneorveiosibbsleermvietdotiinctcheellcsu.ltaunrdes<5% Scellilghmiolnyoulnahyeearltchoynfclelulenmcoenowlearyeerosb.sefrlovaetdinign dteheadcucletlulrs.esanddos=ed15wi%thre1du0c0t0ioung/imnlt.he `ceTlolximcoitnyolwaaysemracnoinffelsuteendocentwehreesolbisdeesrvperdepianrtehdefcruolmtutrheetsreecatueldtuwrietshb7y5t0heg/m. eavvaailluaabtielidtyforfomstphaersceulntuumrbesertrseoatfemdewtiatphha1s2e5s,.25C0h,r5o0m0o.saonmdal75a0beurgr/amtilon(sTawbelree3) No significant `concentrations increase in analyzed. cells with chromosomal aberrations wasobserved at the `The sensitivityofthe cell culture for shown by the increased frequencyof inductionof aberrations chromosomal aberrations inthecells exposed to is nceygcaltoipvheosfoprhaimnidudcei,ntghcehproosimtoisvoemcaolntarboelrargaetnito.nsTuhnedetrestcoanrdtiictlieoniss coofnmseitdaebroeldic. activation. 120 CONCLUSION: `The test anicle, CHO cells with T6358. was and without considered negative for metabolic activation. inducing chromosomal aberrations in 130 REFERENCES; Amitage, P. Statistical York, NY, 1971 Methods in Medical Research, John Wiley & Sons, Inc., New. R`Eevvainse,woH.fJ.:CyCthorloogmyo,s1o2m2a1l-a3b2er1r,ati1o9n6s2.produced by ionizing radiation. International GGioteom.saK-.,stMaaiendiang,oSf.s,iKstaenro,chYr.,omaantiddsS.ugCihyramoam,osT.o: mFaa,ct6o6r:s35i1n-v3o5l9v,ed1i9n7d.ifferential CHV Study No.: 17388-0437 1s 000879 CORNING Hazleton Perry. P. and Wolff, S.: New Giemsa methodforthe differential stainingofsister chromatids. Nature.251:156-158. 1974. Sokal. 1981 R.R.. andRohlf. F.J.: Biometry,Ed. 2. Wh. Freeman and Company. New York. Terasima. T. And Tolmach. L.J.: changes in X-ray divisioneycle. Nature,190:1210-1211, 1961 sensitivity of HeLa cells during the CHV Study No.: 17388-0437 16 000880 CORNING Hazleton 140 EXPERIMENTAL DATA TABLES CHY Sudy No: 17388.0.437 " 000881 `CORNINGHazleton TABLES RANGEFINDING ASSAY FOR ASSESSING TOXICITY. AsmayNo: U3 TralNoil Compound: T6358 Metabolic Activation 59 Dac:OV30% LabNo:CYI206 Treament rOSTVECONTROL Mc NEGATIVE CONTROL McCoy sa SOLVENTCONTROL Wer TEST ARTICLE `Metabolic Activation: =59. ozesem | m1 wn 1 wom | 0 wowm | 0 3 own [0 0 wane [7s son wpm | oom | [C4onSfolluveenncte Convol 5m ww ww ww 0 ow nw " 2 Treament [Scoonlfvueenntce Conmol rosmvEcovTRoL cr Som | ow 1 Ww NEGATIVE CONTROL Mccoys 51 0 ww SOLVENTCONTROL wae oom | 12 ew TEST ARTICLE owe [oss wm wan [12 ww wm [0 1 ww ower | o mcTehaipshaaebsnedepnecovilanstlo.eitAebcdtsuaAlLucplolncovuisnoutafslwtbhesenecvooanaifokunnescewnhbdicanherywvryampieaordvehoeppTohayrcokfosthre+etlaasrchvverdiocfelhdse foc ++0T44o1s0u)mpdeotsapepheaovseefe.sNaoanwailnrygzmead.o+ne=Tocnoclicdeansd dleevdelcelolns.y 9cel viablefo analysis. CHV Study No.: 17388-0437 18 000882 J -- at J wn okra ge 3 SalTleeh lel 5 5 wn 3 Sm g: : ii z i2 cmaSeEo sE sas o co Sen en ea orn -- rer | i = seo]E [ove] 0T sn] i [f cpnro eLn[n ew[| on| CER Abana tome BE Resa Bl rt A } a : SES .:i Em ifi 8 TABLES CORNING Hazleton `CONTROL DATA OF CHROMOSOME9A5BTERHRRAOTUIGOHNS12I9N5CHINESE HAMSTER OVARY CELLS [r---- [Naarmarn -- Rope Conet soe Comet FColnoemoCopnmtn.te _-- Acton se"romorns Fern xNoo oomm: ~ fs wWxe oamo xA isow wwxv oaxe N~o opym w ---- onoo No om wa wwxw oonw W~e o>m w----e oaom x Nne iam ssWoricnans Arion oWracnats avemons >" ooos Ps a5n0 ooos * wwoo wooo no tos a0 io To on B0l 0t0o iin ox 2o0o w"o0 w Eur w nuu CHV Study No.: 17388-0437 2 000885 CORNINGHazleton 15.0 DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS NOT COMPUTED TG Chromatid gap: SG Chromosome gap: UC Uncoiled chromosome: PP Polyploid cell E Endoreduplication: SIMPLE TB Chromatic break SB Chromosome break: DM "Double Minute * fragment: CHV Study No.: 17388-0437 ("tid gap"). An achromatic (unstained) region in one chromatic, the sizeofwhich is equal to or smaller than the widthofachromatic. Theseare noted but not usually included in final totals of `aberratioans they may not al be true breaks. (visochromatid gap, IG"). Sa aschmromaetid gap buta thesame locus in both sister chromatids. Failureof chromatin packing. Probably nota true aberration. Acell containing multiple copiesofthe haploid `n(un)ofmchrbomoesomres. Not counitnethde cells scored for aberrations. ahansceflailleidn. whNiocthcsoeupnatreadtiionnothfe cchelrlosmsocsoroemdefpoarirs. aberrations. An achromatic region in one chromatid, larger tfhraangmtehnetwmidatyhobfe paarcthiraolmlaytiodr.coTmphleeatsesloyciated displaced. `Cahbrnoormmoaslo(mdeelehtedaa)cclhseraromborseaokm,feowrimtihnagnaancentric fragment that is dislocated. t`Tehremsienaalredeslmeatlilondsoaunbldesdoomtse, isntoemrsetoitfiawlhdieclehtiaroen:s `and probably small rings. Theiroriginsare not distinguishable. 2 000886 COMPLEX ID Interstitial deletion: TR Triradial QR Quadri radial: D Dicenric: DF TC Tricentric: QC Quadricentic: PC Penucentric: HC Hexacentric: R Ring RC Ring Chromatid: RF CHV Study No.: 17388-0437 `CORNING Hazleton Lengthof chromatid "cut out" from midregion of a chromatid resulting in a small fragmentofring. Iying beside a shortened chromatidof a gap in the chromatid. `Acnhreoxmcohsaonmgee abnedtwaeneanctenwtoricchfrroamgomseonmt,esw.hiorchone results in a three-armed configuration. As tiradial, but resulting in a four-armed configuration. rAensueltxschinanagcehbreotmwoeseonmtewowicthhrtowmoocseonmterosmewrheisc.h This is often associated with in which case it is classified an acentric as DF. fragment Dicentric with fragment. An exchange between two chromosomes which results in a chromosome with three centromeres. Often associated with two to three acentric fragment (AF). Such exchanges can involve many chromosomes and are named as follows: four centromeres, up tofour AF five centromeres,upto five AF six centromeres, up to six AF A chromosome which forms acircle `centromere. This is ofien associated containing withan a centric RE. fragment in which case it is classed as Single chromatid ring (centric). Ring with associated acentric fragment 2 000887 Cl Chromosome Intrachange: T Translocation AB OTHER GT `CORNING Hazleton Exchange within a chromosome: .g.,a ring that does not include the entire chromosome. `Obvious transferof material between wo chromosomes resulting in two abnormal chromosomes. When identifiable.scoredas "T" not "24b." Abnormal monocentric chromosome. This isa cthherkoamroysotoympee,whaondseofmtoernpthhoelroegsuyltisoafbanormal for translocation, pericentric inversion. etc Claasscsriifbiecdattoi;on.8.u,asreedciifparborcoramlatlriatnyslcoacnatniootn.be: AA hceelalviwlhyicdhamcaongteadincselmlosrheoutlhdanbe10anaableyrrzaetdiotnos. identify actually the typesofaberrations have>10,e.g., multiple and may not fragments such as those found associated with a tricentric. CHV Study No.: 17388-0437 2 000588 GPaRvOTSOCTOILXNon.o. 437, EDITION 13 CORNINGHazleton CHOWOSOMAL AoBvEaRrRyAT(IcOaNoS) TcNriCAsDNISE BANSTER LcGhaoebronwrioanrtgokrHyIansiPeprtraocontgirceIesnsc.(aGnL(dPG)Vt)hReewgiufllialntailcoonrnsed.puocreThtwihsiilslprsbotteuodtysaulbi,jneccctroimtptilociaaalnucdeipthwaisbyec(hsQ)uGaoloodifty ACsosnudruacntceed biyn aCHcVcoratdan3c3e00wiLtehesbSuOrPgs aFtikeC.ornViienngnaH.azlVeitrogniniIenc.7210T2h.e study will be PART 1. SPONSOR INFORMATION AND APPROVALS I. spowson TomvTIEIGATION Conpany vase: _BM ee------------ Address: -- 5tA . d Lm N 00000000 mm. pEstemmmceonon: 7- 358 IIL. TESTARTICLEAKAYSIS DcofheacretachecneienSrapitosnitsooinrc.sofasthedefeisntedairntictlhee GsLtFabiTleipuciyatainodnstheistiehset arretsipcolnesibility IV. HOTIPOFRIecUCLAATORTY SITBGOSSINON mIsnusbtomirsdbseeironnot.ocifciCoeHmdVplymiafiwniatclalhinotsrhe4paGrmLePascorefergaulsasctthiueoddnyusl,eis ocfoinnsstuteluntddiienedgs fwolhraibcorhreagtuaollraliteosruynder CCheegulraetsuolrtysreovfietwh.is sPlteuadsye indicate vhich agency. Lf any mighe receive = ndecernined o--d ron L--_om eatsen -- od eea.riema 2 wer TZ ow Tome C0 om ass 11 000889 Protocol Mo. 437, Bastion 15 `CORNINGHazleton v. stor ares Proposed Expecimental Sart Date -- ss Proposed Experimental Termination Dace: ~r ~ t------ VI. APPOPRSTOUDYVFRA oTocLOL Stugy bireccor: TVs ne Sponsor Soe Coe Bases Dace: _2/1nfps-- ass zoe 000890 v Protocol No. 437, Edition 15 CORNINGHazleton PART 2 STUDY PROTOCOL CHROHOSOMAL AOBVEARRRYAT(ICOBNOS) ICNELCLHSINESE EAMSTER 1 ommerve Tt(heCesHtO)oabcrjetelicltcsilveewittoohf iantndhduicsweiitcnhhorvuoitmcorsaoonmaaselsxaoygaebneioerursattomieoetnvasablouilanitceChaitcnhteeisveaabthiilaoimntsytesryofscoevama.cy mn. perros SsSuatttraeutcciitoaunlrsa:landacahnacynagnecsbaercaianndnoaglreyenzasecdracnainugseesceunclthtsruormerdoessoucmlealtllsf.draommaCgdhearmoamagonedsomctaholrotmhboerseogamkeasnletairce cthhaenrgeefsoreha&verebleeevnanatssotcesitatefdorwipcohtenctainacler.mutaCghernosmosaonmdalcaracbienrorgaetniso.ns are dNarueemteercnitotceadlsicnaorbaeendrraaitsnisoaanyscosnuotcrihnvuaaosruisatthiicosenlslinlfwirnhoeimchtshuecchelnloasrsmaCplHaOsscchertlohlmrsoo,suognhasnldonlanyruemboeanroet division cycle before analysis. ChotnrheeomcsohasrmooemmaoLlsoocsmuhese.,ccoarcitohne:edxacmhaanggeeebxeptrweseseendbointhbocthhromsaitsitdesr cofhrmomeaetidtshanat ChBretowmeaetnidcshrhoemrartaitdis.on: damage seen in a single chromatid, or exchange III. RaTIoMALE ciTahnneducaiesndstaeycLaecsaeiiomwnssicthmoayceesltrlaesbsullittsoh iinwnhdebutrceehaekrcshrtohimneosctohemrsatolmaatbrirteniackltseh.atoraCrhieetmsiecmiatelhtleayrbolites svFieesppiaabirlzeedpdraobmcyaegstesh.eescsAeulblcehrziantthiastouncshbraeaarwkesay4docasonnostteoqurbeeenjcoueinndeoftoefcftaraiebljluoerienofoirnFbmesinesoitrtamkaeilsn in configurations (reviewed by Evans. 1962) PAarfbotecererrsastcihoeonmrsicbaealrecoenexvpxeoasrmutirenede,divnbhteeofnocreocmepltllhesexy ecdnaetnreirvbametiitlvooesssitsdduufrroriinnggthesthuefbisrdesiqtvueinsttiiomencell eycles. In the case of the CHO cells used here, most dividing ars sof 18 000891 Protocol No. 437. Baition 13 CORNINGHazleton stChieiiolsnlcsedtlehellrxsaoa)yum.gihanneHddtohwae8elvlcteoeorwl,l1f2mcoayrhncoyluseri,stcewseaatrnfdtaegrrrttohivitcisrlheeaassotfsmcaeaydnuatsmheaaagsreseedbveieecncneeltldhdseeeislriagynFeiodrfsttpormoiogtororesesi:rs in the time becween trescment and cell fixation (see Sectbyionadiovast)mencs vo mmm A Zestcells hoSTaarhmniesgtiFCenrHraaO.nlclciyeslCcloeolb.ltlsaiinTnetheoedwebsfceerlodulmseserDdirvv.eeirdne5.fthrsWiouosmblsfaaefsqn.sueaonyUvtnalir(yviCeaHnrsOes-ciWbBltiLoyo)npesodfyaceoicnfaal.ifsoCurhbsieinisee:smee jRLsoaube8osrapwteiortrmhyaneatnotmomdcaaeillnltcahiLrnionmekoaswrciyatoehtynpauinmcbaevsretraabogfieli2t1c,yy.cle ThteimeCHoOf-WB15Lthvisosub1c0lone 5. cell Culture conditions wYCpAieHLtnOLhiccieablelpllpisirnnocxwuii(bln1ala0t0teebdlueynwigitr1st0o/h2wanlLf)oeiotnsaaelnMdccCcaoaspylts'frsepisnteSoraummyc,cuilntLuorg(el10u0cmaenmdiignu/emml(s)uap)pGl.uelmceunrteesd 37C 22C in an atmosphere of St + 1.35%huCmOipdiifniesdir.incubator or : ZestFortveoplasmsContamination HoHSyvyececaoroippnlletadahsesaewssciycthatorepeHslotadeisecnmthgisectcitse3cdp3e2eg5ari8sfonorsammnofeadldlot.hbresoeiurcntvetileenldnessl.eyulnydbeyrfelOxuVoarmmeiisnccaretonistocnosponyfa.cksslides b2(e0SdectafiproiondmlyV3 .d3ree.cc2ro.eg6an0si)ezedd upbtoatkhe forfomBrcdheUrdfluionretshecAennycte.lclsoemcatyiaenmreiednsadtopiiponengarwaoenueclred . Racvocrpesubilicy KPSseuaoabribsynieolqtiuyocefpyne,cclhsyectealasblcsisoslraiwetydiylilsiinscbeulyintqdouuegsireeddnectntoiihnctasrctoaagnhnetaanlv.yesscirbsue.teinnycTolobneeecndasuurs(eeetosthasemreiynnd.and idSioLsnLcear3bde-e6dtthaarfvatenedsrfepraesbroiuoidtnicecaiulgllhtytu,reverebekecsuflotrofuereccdroyntotipTenrhiueecosseueesravsawtcteouieclorktn.su.reha.nadvSeamwmaideers ass cof 10 000892 Protocol No. 437. Edition 13 CORNINGHazleton E. less aruicle Stfroeelseitdacruotlritcullrieequmiiesddidtuiemss,tsovlaavrtetedirc,liensdainmaerteahpypslruoipstruailbaflteoerifdoserolvt(eDhnMitSsO)as.suscashyc.ahsazeTslhe,eruemr acdcueelsttiournreeed. afeidSniaeulrmi,aclonucdneilnletusrstaitoiinnossnosvliulbblyliiabetdydictraeirqrouinierdoefsou0t.405sloaralgae3rpefrvool5aucmmheli.eveThe mumenadlxieiusmnsu,moutfphineartlowis1C0eo1ncsewpanettceriraf,tiieod1n1sbyDoHfStOhesoorSlpvoeenntsthoarni.onl.utphoertco0u.l-3t810u7reaccewutiloltnluer.ebe, The test compound solution will be prepared immediately before use. F. control Articles 3. NegandaSoltvenitCov ntroels Hciosnhtoauitnacotnliyvacteilolns: andNecgualttiuvree cmoendtiruoml.s be cultures containing the solvent for wiSlollvebnetccuolntturroelssvwhiilclh the test article at the same concentration used in test cultures. Haaib5otvtheha.ectwtiievlsatltibcoeunl:tturreeNaste.egdatwiivtehan$d9 ascotlivveanttiocnonBtrioxlsi,n tashedesfaimenedvay 2. Posisive concrols aoKtnneowtnwcoomnuccteoanngtceernnaittcriaotnainwodinlsclhrtoobemeocnshsooumsreeenbardefeaoqrkuiinatgbeerargraeetsniutiostnswsidlnlabluytbseioeun:lsyed Wdiiontehswoautnteoartctarinedqvuaiutrsieeodnm:eactabMoiiltmiaoclmyccaicontnicveCantt(ir4oant).ionisMsICaofwci0ll.al2stobtegoen0d.i5stshonagtl/vmeld i8n-12thoeurtasn,geafnidngi0n.g040asstaoy,0.100.05 wtgo/a1l.0fourglallatefrorfifxiartaicoino.n st imbtiechrcooasncoveneiervsta.esdioAtnno: aexcMataminpvlyeesiuincscaegrcemynecsdliodaptoheossnpohtbaymaicsdtneidymi(erCseP)c.tfloyuunsdebdutinhemruest ma8ne5dcacbuhoseeldipcoastiatcitfviienvaaltcioocnntornocsleynsttteroma.tdieomnoCsPnstowrfilalt1e0 bettohedi3s0ascoutglilvvaeilcdy iinnofathtteheer 53 hroanugse.finandding10astsoay2,5 w1g0/atlo f30orulgaatlerfofrixaftiixoant.ion ac 8 to 12 aos 5 of 18 000893 Protocol No. 437, Edition 15 CORNINGHazleton . IheMetabolic ActivacionSrsten etTinheoznymien(sNAvDaiPntdraonadmneteiansbeoorclgiityrcipcraocdatcuiicvdia)n.tgionsEynssztyysemtmeesmneaccreoesnssaicrsotynstafoiornfedrtahteni'rLaivFearc. AscPuhrtceehtmpoiasAczearalotscoirloontrionomf1op2r3ole4itvaaetscorstiimiuvincedrcuohcfsloeroomrmesien.dsceym(eT5sh9e cfSaropaacbftlrieaocnt)oifofntr.roamnpsrrfaeotprsimeirsncsgeianted abiepsprtpohuxarvicemhdaacseielmdymefd-ri8oa0m'tCeclouymnecbrieclfioaursele.ussuepAplalfinirqdeoucmosatdmsdaaelnodedf itStosphriarasegtu`$ace9oirnbefea.rdcaicpeftoyrinooosrnoeicnwrai,salcl mixture to form the activation system descrives berow: AIsDoPcic(rsiocdiaucmidsale) Homogenate (59 fraction) 2L.s7 mmgpliaall ((110..58mm)n) 1520 wl/ale, Vv. Eempoma pssion A. SoluandbDosieDleteirmintatiyon T[haekinngaxiinatuon Sienaftoerdmavciiloln adocsceounwtillbotbhe abveai5laagb/lael,on dtsheoetleurbtmeisiltnietadyrtaoinncdlaea.ncyaserTheelbeyvhacinagtsheecrybsasosietseormicicy DtSepLse-tnsleuorng.lesssAeriareasdnogseweilolfrandbgeoesecuseinsslcecessodspveecrhiiniifngightehederfivbdpeyorseotelhsriedmeiarSnrpsaeornyossfporre.scaianfggiaeeiidnvdubisyngtchrea dpSeroielolunibinizilenidacrywyaitsneofrlo,urbmiaslteiirtouynm-fhtraeesset bcweuiellntluprbreeovmciaderedrdiiuebmdy pcetone. Stock solutions the Sponior.Ifaeo Douuro.wicshthsaeneerrileor hpsruoenlcduirtpeiiodtn-saftoelidnteaiscnte.tcounletThuverielslmolevdbieiennutdnDiMlsSiuenOtleeaodcrtpe1erd:ce2hc0wai0inploilltiantbweeiclultlsehteeubrre.e amieisdaeiteoerns one in a Bives the best solubility after dilution in mediee. on cuimn vIUSoasefs,oeSrssrsmeduaumnnaocyengiotnifmleot5.th9se,haEos$ams9coahgyae.tnlaovttaeBroeifcpoaeuurSssoechuolcmhteougreepnraowctielelinisdceoprneetnsedenetdonwohsehne85 lphoootrscmiopfieans$ys3eein GSSaaimf%eeccrtteiynoicnneitocrfhoe|smeincriasnleesr.,chsrTuohcmehatoaispdticemyxcuclmhocap$onh5ngocescepsonhntacirmneaintdCtieHro,Onastcibeoeilnnslzso1r,isens)csepedyiercearneegd.nonbased on S945 used in "19s subrequent assays with that particelaendloenhisoeaomgen of of 1s 000894 Protocol Ho. 437, Edition 15 CORNINGHazleton vtIenhhaitscolamereeccachsooemsspe.antiatbneldsetthwaeirtthhiicgltheiesssstuaecdeocsuaelptputararese.inetldyIwniilnSlsuochlbuebclatesheasti,nintshowelhviencths an evenly dispersed suspension can be prepared. asTsphseeacyitfoiupcAldlgoesaebveite5ye.sntge/dmSlof.lourvbhilliiicqthuyidsatyetsestatilnsgoarwbtieilcllceasblecuilntuhettehdesamrfeaonsagseftihending described above 5: Rangande Detefrminiatin onod fFisisionn Tg ine i. pastomle dHTaoemsqatugieziemioanwtnopeccrhiheomrdiocmaoolsfomDcelAaasltstoyegnretanhtseisoi(nscshtrvooimscoiosbnolvmeeertabcreimanikittioinsagilsa.DgeUnAtHsa)ny cpchreeolcclmseosssboensaeurcincpgersespafabuerlrarltayicoinaonndisssamtyahyusdineos.telceocTmtheepdlectiteoneatlohfleowfdiicxveailtsliisoonntoforgo acyfhctrleoerughotfreDGaNtAHmOe-snVytBn.LthecseSilislnsce(iSsu-npadhbeaorseu)tnora1mn3adlhoruceriasrc,chumftsihtexaantfciieorsnst8tmheittoocse1il1sl htohuecsfirasfttermitaodsdiistioanfteorf many clastogens inhibit tptheresotgbraeergstisinincoilnnegofwioflcleltlrcseoaltltmehecrntotu.gchellHthsoew.eivner. Amcsiytcolaesn.isalatwneidlslndaatnmioavtgeedbetoccelocllaslrercyvtihenidgchoaturteacnosvee8vr-er1a1elnohuotguehrstsftoiixnaptrwiohoginrcehstsimet.o cicenelglllascya"ccpleeeakf"diexleofadyaavtbielr4lcastebieriomensadyeioeflbdyt,idmeiasffpeirrneelniocmriidanelarrysttoAaisansvieensigsd.memnsts.of PbCAerLloElmso-of2vidlaelocxhbreyoumgrorisodowimnneein(s(it3hsretdeUrcpdr)ce:hsreonamcfaetteirodf)twtwohielcleblalbseecsyaocnrlaeelso,ghueeaovnie5ls-y tfsheuerbesntsctieatiuntC.eaduseTwshieththcehBarrcdahUcrrtodemratithsiatdnisctthsoetdaoiitnfheifrne.gr inThitshechreemaicctailondwiifth paciern of a cell tcthehelaltsc)ohmaspilseutanedecrllgeeoannrgeehditofwffoerecevoneartpyilaetctiheornocmeolbsleotmwceey.ecnlescInhwriotmhteahtiCBdHrsOa-UVesEdlLon(gcMe;ll oesnxyepstoecsaeu.lrleacptypocrloBexr,iamUacetherlosymhaotw8i3d1ts-hi1s0a0r2eHy eosfvteancilenylilnssgeafpiianxteecddera(nfH.,tecreAlf2lt6se)rh.oounrlsy cAihfnrtcoeemrromsseoodmrieeastetahcpaenatdtoienefrfnescryecanclceeiabfluotluyndlessicsnaiwnthehidacnh(Htsow)no.lycomp`paTlrhecusst,eoifcfycttlhheees. ass 70f 18 000595 Protocol ass No. 437, Edition 15 CORNINGHazleton Mcierllceclylcslewiilsl salpopveeadr dionwnthbey ptrheepatreasttioanrstiscaldee, abfoctehcby36ahnodurs iben mBarddeUcdf.ronAntheestfirmeaqtueencoifestheofdegrceeellosf. ceTlhle cfyicxlaetidoenlatyimmeay sfoerlecttheedstuobseeqnuseunrte cthhartommoosrotmeCealblesrraacteionintetshteirvilflirstthemnecbaeCPehlalsse atfotetrhetdrievatimseinotn, staalgleoving the progression of damaged 2. PreliainacyTestfocToxicityand GellCrcleDelay Cpullatsutriecsfalcaesksseiende5d aalt oafbouftres0h.3cuxlt1u0%recemleldsiump.er 2o5necafllask wSiplolnsobre irneiqtuieastteedd dfoosrese,achsoltvreenatt,menntega(t1i0vedoasendlpevoeslistivoef the controls). a. Zestwithoutsecabolicactivation wTihleldabey aadftdeerd ctuolttuhreecuilntiutrieast.ion,Incsuoblavecnitonoratteasbtoutart3i7cCle W(iilnlalbeccoonncteinntureadtiofnor1a0bkoMu)t w2i.l0ltobe2.a5ddehdourasndvhtehne cBurla-vrd acbuoruets v2i5l.l5 tbeo 2c6e.c5urnheodurstobethfeoreincfuixbaattioorn.forA4pprootxailmatoefly W2i.l0l tboe 2.v5ashhoeudrswitbhefoprheospfhiaxtaetiobnu,ffecrheedceslallinmeonotloayreermsove cwohnetatiensst Caorlticcelnei.ddaantd fresh medium will be 0.1 ug/ml and 3rdUsd added at 10 which ui. 2I0n0cuhboautsieo.n with Colcemid will continue for about 1.0 to b. Zest wich che setabolic activation svsten Obrnueefmfodeveradeydaanfsdtaelritnheeculctoetlulrrseemwoiivnleiltisabecteuinov.na,sheCcdeullltosunrceveilwslietdhichuenpnhwoisblpelhatbee eoifndciutubhneicwetidetshtaotuatracbiofcuelttea,l37ctChaelffo5r9searrbueomauctt(iFC2oSn)h.omuitxstTuhriene.sthhoaerntdprgiernsocevunbicanemtciiaxoptniudrlteyineaitsist37oxuCis.cedtSobeerccueamlulssies: poaraloisltooenegedendctyomeexapvaoosciutdrievpioctsyosicibshleelo$9st ilinvaecdt,ivahtiigohnl,y bryeacbtiinvdeingintteormseedriuamtepsrotperiondsu.cedofbysho$9riAenlzlymbse.vasAhfetderacthelea2sthoutrwiceexpwoistuhrebpuefrfieorde,d stahleincee.llsand ote 000896 Protocol No. 437, Edition 13 CORNINGHazleton WBiolrlmalbegardodveedh.medIinucmubcaotnitoaniwniilnlg b1e02coFnCStinaunedd10forknabBorudtUrd t2h5e.5latsot261..50 htoour2s.,0 hvoiutrhsCooflceinnicdubat(i0o.n1 ug/al) added for c. Sumacy of Treatment Scheduler sooac oms) Ist ome) 8waa FE we ab: ged ue 8ows; Glee aaEs fede HaEsIsReSs "Ties given are ssoronieste and my vary according o the ze of th assay. d. Mloediefripcagtionfortestacuicles thatarevolstileand/or ctFahopersctuuelnstttiulraerttihfecllaesaskrsciccvhlitellairsbeevaiksnnhocewudnbiottfoefd.bweimtTahorkcteoidmglphyetnlsyvaoltcaeltoisfleoedr, cLeanctkraotfioCn0); cwiirlclulbaetiionnc,luHdEePdESinbutfhfeermed(i25un.mi final con siIefndcimluaumrd)keeddduirnpiHntghcehatZnheegdesisuonalcuebtoilniaotttytiecmetpdetst(,Lio.HecE.oPuESnctoelbrourafcftecrhatwnhieglelspif bien v(cihalaplnpgreob.xeimpaFetucerflotyrhmeer6d.8netuotocmeaa7il.n4i)tzaaiotnniloynnoar(fmsat.le5.r.ccuwlotinuscruheltHCaplHtioronranwgNieatOHh) che' Sponsor. BeBdreofvmoiercheroifsnichxoiaptbieiotnit,oonefaslntaiskmmsaittevoitlcilyctobtesouxepixpcarimetsiysnieodenx:purnedesOrsbesdearnvaasitnivoenrs:. msoifutcofttahicece dcoeefglrletsehe o(mfloancrogoeln,atyleurreonucnodyredfa.nldoahtaeipanptgatrheyinntcetlhalevsamiloeandbiitulhmeitywilolf 2Lbeevlemolawdeere.xdpoescCeteeldllsevtweolisly.lielbMdeetahanapyrhvamesesetteadpchealflsrseosmwaitnlhdle fhirgohmesatc ldeoasset be collected aLbyceindmiwti(ot3t:hi1.c75vsiohva)k,eKC-ldo,friopfpi(exTdeedroanistnionmaegclahanasdsnoTlos:llmisdgcelhsa,,ciaa1ln9d61a)ac.iertiscvol- dried. ss 5 of 18 000897 Protocol No. 437, Edicion 15 CORNINGHazleton DWfOilLfuEfofec,reesncte"1in9ac7le4;-pslGtouatsio-n,Giingeemtsiasal.a(.cFFcG1o)9m7p8lt)ie.schhendiSqiubieydesa(awmfiotldelirfiPbeeedrcy and ss$a5tmaeuingeb/dulf)ffeorri,natapnhdolseepahxsatptoes1e0dbmuaitfneurt5e5s(-p6iw5i6tC.h8t)So.oe"mcbohlesatnctke3.d3l2i5gi8nht:the dFiirnfoafmlelr1ye5.ntvisaalctidieotsnubwebiseltlufeoarbne tchshetraoiamnmeaodtuindvtsitohf(35.t12i0mGemieirmnesqsautierfreo.rs for 3 to 20 minutes and sic dried. Tbeheexsalmiidnesed frfoarn will be made to stpchraeensheainngcdheescotflasddsoeislfeayyeLdoenveeclelshlusn".dhraerdAvnemsesttceardpehmapwseiell cells as Hy. Hy + or 3 Hy 3: RTeetsetmminasionof Poseand FixationTimefor theAberration mTvaahnaxectiei)oodnuosnsaensddoofscseeetllolesxceitlcceeiydctctyleefdo(rdcweoiltnlahflyelueabfensrcsoeamthyetevhdhieuilcglpthrieeboseltninbiaansnedadcyceorntlosteah:eppoaebnnsgrert.he at0hneal11yazbashbeonluceresgeoacffatpeorhbusseaerdvdeicdteilolcnselloafreccyehclexempieccdaetlleadyw,itloldaobesbeeixshawaecitseiiqown,nheidc,shiInreaI3n TtS1ui8EomLehLdoiutcfroyfscelgreioevrnedte22lfanyihixonauiitcsoisuononwmbialoetlfrivneeb5dse0.7vsic&elelllellcastteedrin bfMaiysxeaadtCiootnnhetthiTemoe smoestusicmhmaeroresed 4 the top dose causes delay but thbee luosveedridfosneescedsosarmyac)(.op, . Chromoscne AberrationTest Cpffiliuvanlesdttiuinrdcgeossetfswelsiawtlsilkl(libSneebcet1si0eouensadeleddV.3o)fa.tdefpareebnsohduitncgu1l.ot0nurte0them1e.dr5iesaxu,i1t0sAcoefmlilntsiheopnsreaonf7g5e.ent CobhsaenrvBaatlieo-n1so.g diluzions dependTihnegdoosnesthemaypatbetemronreofclfoosxeileylcypieced wDtiuolplldiocbsaeetseuseofdflasfpkoosrsitwsiiovllelvencbtoentaurnsodeldncefogomarptoieuvanecdhsc.odnotsreollsevealn.d foSringSalcehfolfasks 4195 20 of 18 000898 Protocol "9s Mo. 437. Bdition 15 CORNINGHazleton 3. Assaywithoutthe mecabolicsceivasionsvsten O"tnheeepldtaaecyesdtavfaitrtteirhclcferu.elsthurTemheediintueismttiaatanidrotnit,chelethcweeilllclsulvtbieulrlelemfbteeditinuremacvtuielldtluwreibsceh foxr0nthteheenptrierleimipneariroyd,testthe(luesnugsclhlyof8 wtohic3h0 whiolurls)b.e dAebtoeurtmin2e.d0 bhouutrtserebdefosraelinfei,xatainodn,frecsuhltmureedsiumvivllillbe bveasahdeddedwhitihchphcoosnpthaaitnes CCoollcceenmiidds h(e0l.p1sugt/omlaccfuimnuallatceonctehentrdaitviiodni)n.g cePlrless.ence of 2.Assaywishshe secsbolicsceivation systen Osneemovdeady aanfdterthecucletlulrse wiinliltiabteiovna,shetdheocncueltwuriechmesdaliiunme.villThebe cBiernlolvtshtehwimplerldeisuetnahceewnitobhfeoutithnecufbetateatsletdacraatltifcalbseoe,urtum5937(FrCCeSa)f.cotrioanAbfomtuietxrtu2trhheeo,u2rasnd p1R0oh%uosspFhCeSaxtpewoislublrueffbeepreearddidoedsd.a.licneelI,lnscauwnbidalclnioonrbmewailwlalsghreobdveehactonmtelideniausuetmd ctwfooinrcteai&nwiintgh 2f(.0u0.r3thhoueugrc/msplertiofoidnaaclcofumcuoalnpacpetrneotxritashtaeitoednli)yvidpirnetgsoenc2t6elldhsouurrisngwitthhe Claosltcen1i.0dto 3. cselcl ainanidscnorigng MnsoeettcatpibohenasheVa.rBvc.ees2lt.leesd..wilTlThhosebeesflsliuadbsesksesqvuiwelinltthlybeecxaocilerlsescidtvreeicedtaonarndidciftisyxteadwiinlealds in wzsi7ltlihedneesGiaweminsldal.mobuenAtcfeotddeerdpderrfymoiarnngea.nntalltyyh.seissFleoirxdcecesopntvtirlfololr boetfhecbiipnasoses,ditiitvnhee acboenrtrroaltsi,onwhfircehquweinlcly bise acdheeqcukaetde.first Go make sure the Nctohhreemadtluolppyl,ifcoautrtweo dohfsulenasds.kcse)d,Ifceilftlwsoavahpiuelnradbrdleoeds.e cvelilellvlselvbee(r1e0r0eandoftrfoarmvoameialcshaabclhoef of h2u0n3dreedachCelolfs twhielsle solvent controls. dboeserse.adlofvreorm edaoscehsofmaythebeneegvaatliuvateeda.nd One Ac least twenty-five cells will be read Bcforomenpalteeotrnee thnpaounsaibteSir0v2eofofco1cn0e0tlrlocslellvasnidthpefcornoemcuoltrthuormseeoreTcauylatbuneorrtersatbietohnaastv.aihlavaTebhele due to toxicity or quality of preparation. Cells will be 1 of 18 000899 Protocol No. VL mm 437, Edition 15 CORNINGHazleton S2See1lll2pse2esvsi(tchafnogrcehesocfnourm1ib9negrtoonof23t)cheewnitblralosmisebreeosafnagsiqoyuteaeldm.coorphtohelogmya,dandcuomnsl]y L7Sbioetstaaterndidnoafgrsodaabntfehorerarmbascetecilrwolaintlsailoynsb.ceobretuehsderedemalniotdccoraottrsehecdeciaornirfeddnesaeftnciaaenglsieysesaieTrosyo..catiFooThnre.eerearcoyhpibceoeclel to this protocol (pages 16-10). nt is seeooiiy A DPatra esentanagtsvailusotinon DiPe3aor8taaicayenzwldeildl,.lisabtoneycdphesospumemacoatfccieidnaztbeasegadrepcssaitnwioifotlnalscbellebflesosunnsdobh,teoeavdrifinrngbegeqtutaehnbeceirneursmabteoirfosansborfeEceaetlcilioscnns Eirnuteo tbeheeskKaO.TALL for abezcation assessment sinwciellthseayr mnaeyscchortyse AssayAcceptancecriteria dfFnre2si,uvvlaiesStsaniyAoeonpinellnyalanedinfbsneaaunlcnlaoicnttossifivdaaetntrhdieeodnfaoLsclleclceotbpweitioenndgrsepcfeorofnirtteterhvieaalsusabrtesiromensatcoifsointnessset.iey needed. to satisty the acceptance cribeseed indepentonniy oe * tasacistacsory conceals The assay will be repeated if, I+ 4M(P4ePpepcenseLgLiaimtmiiitvteooSfrIthteohre rmasonorgleevenooftf hciosnttorroilcails cells with mscbooernrerroettrhiaonmnarttswe.irce the 2 aT{Idfh2ee5qt,uhp0aeo:ts0ept1oi)sivnietciahvnacenoanstctsrohanoeytlrpovoclhoeelsreruedelscsunlce4ttgsaitnniovtethseatngdsneistssiotcwvaenincrslySchooiiigrrheerrs. 2Isieoh2enm3sa7icssttciitvivaivactateyciioronoufnnaspcsooasnyictuizfvazeielnscc.olnyttrhboeultitvdeateshcmteioonhnpseoeesardinasdrnieovnsteonesbmenaeensrieenerttattocnnna'tlhte the cells. "rss 12 of 18 01 103675 000500 Protocol No. 437, Edition 15 CORNINGHazleton Leck of Toricicy Isfigtnhiefiacabnetrrarteidounctiroensulitns caorneflnueengcaetivoer adnedlaytheirneceilsl mocycle hpirgohgerressdioosne,s tchaen tbeestacmhaiyevebde (rie.pee.atneodtatLihmiigtheedr bydosseoslubLiflity or not greater than 5 mg/ml). ExcessToxicity Tahtelaeassstaytwhirlele bdeoserepleevaetlesd. if results ace not available for Sporadic Increase LIefvealssigbnutifincoatntin icnocnrseeacseutiivse sdeoesnesatanodne ifor tmhoerree diossneo cbelearrepeeavtieddencteo vfeorrifayptohseitisviegndiofsiecanrcees.ponse, the assay will Isfulttsh,e aosnslayy thharsee tdoosbee rleevpeelastedwilblecabueseanaolfyzeeqdu.ivoicnasltearde-of ouc, in the second assay. C. Assay Evalussion oTfhe tfheolltoeswtingartfiacclteorsdataar:e taken into account in evaluation Overall aberration frequencies. Percentage of cells with aberrations. Percentage of cells with more than one aberration. wdSiosvteih,decnoicm.ep.l.efxoraadibnoecsrrreeastrsieiolnnagstedammaoyiunncbterseacsooefnsdiiadnmearagebederwrtiaottihhoanvisen.crmeoarCseeilnlgs significance than sizple aberrations cSotmaptaicsetictahle apnearlcyesnitsageempolfoycselltshewiFtihshearb'esrraExtaicotnsTeisnt crteosted ciceonlncltrsreoalswsiintgh(SonpkuoamollbeedrandorfeRsocuhelLltEls.s fwr1io9tm8h1)s.aolbveerAnrtaltiianonendasrnwetigratethnidvietnecsrteasofing ddoisfefer(eAnrcmeitaigse,con1s97i1d)erewdillsiaglnsioficbeantpewrhfeonrmepd<.d.01T.he The sciinealntiefviacluajtuidognmenotf the test article will be based upon "iss 13 of 18 000901 Protocol No. 437. Edition 15 CORNINGHazleton vil. sermeces 1. SAornmsi,tagTea.c.P.NewStYaotriks,ticWaY.l M1e9t71h.ods in Medical Research, Joba Wiley 2. tEivoann.s", I8n.t0e.rnatCihoenoamlosoRmeavliewabeofrraCtytioolnosgyp,ro1d3u:c2e2d1-b3y21i.on1iz5i6nsg radia. 3. Gi6no:t3do5i1f-fX3.e3,r5e,nMtaie1ad57al8,.GiS.e.nsaK-ansot,ainYi..nganofd sSiusgtieyrm.chrTo.:msciFdaec.tors Cfhaevmoslsvoemds, : Psetrariyn,ingP.ofandsiWsOtLeErE,chr5o.matiNdesv. GiNemastaurmeet2h3o3d+1f5o6r-1t5h8e. d1i3f5f0e.rential S. Sokal. RR. and Rohlf, F.J.: Blometcy. Freeman, 1981. HTeelrsascieml,ls7.durainndgTtohleaacdhiviLs.Ji.o:n cyCchlaen.gesNaitnurXee.ra1y90s1e1n2s1i0t-i1v7i1t1y, of1961. VIII. REPORTFORUI CreHporetmpwliolyls aprosvtiadnedartdhesefpoolrltovifnogrmaitnfofromrateiaocnh. assay design. The Final QSSuptaoalntisetomyrentAisdsoeufnrtaGinLfcPiecCaotsmicpoalntieamnecnet dTSeeissgtcnraiatpcuttreiicolnoefofsitdtuehdneytidftiierscteacttaiorortn.icalned included in this section: aCnHdV dSattuedyofNumrbeecre.iptAvipihiysibecal DSTatytupedesyofodfizaesscsttauodyryaanindnditpisraeotntioiocoonrlatnneducmhbnceiorcm.ipalne:tion. oo MIEenvttahelorudpasrt.ieotnaticorniteofriaresulcs o CReofnecrleunscieosn.s Test tesults presented in tabular form. IX. CHAONRRGEVIESIOSNS 4#217gnecdhanbygescheor StruedvyisiDoincsectoofr.thdiasteadp,proanvdedmapirnottoacionledwvililthbethidsocupmreonrteeedcd,. "rss 16 of 18 000302  Protocol No. 437, Edition 15 `CORNINGHazleton X. REC70BOEMR AIND TAIS NED AgLeLnerraatveddataas, adorceusmuelnttaotfiotnh,is resctourddys,wilplrotboecoalrsc,hivaendd final reports in the storage fsaucbialiistsiieosn ooff CthoernifnignalHazrleeptoornt Itnoc.theforspoatnsolre.astAfotneeryetahre fonoelloyewairng rpeetriaoidn.edtihne tshpeonssocrormagaey felaeccitlittioeshavofe CtohernaifnogrHeamzelnettioonnedIncm.atefrorialasn additional period the sponsor. of time or sent to a storage facility designated by ig 15 of 18 000903 Protocol No. 437. Edicion 15 `CORNING Hazleton DEFINITIONS OF CHROMOSOMAL ABERRATIONS FOR GIBMSA STAINED Criss HoTcoupuTED To chromatid caps 5 chromosome Gap: Ue Uncoiled chromosome: B bolyploid cell: E Esdoreduplication: i(0n76ao1nrdesgcmaahple)lo.emractAdnh.anacChhterheomswaiitzdeitchof(oufwnhsiaecahcihnieiedsm)seiqriueatg.tion aFThi1enbsaeleatcToertuaenlsotbeordfeaabkbusetrrnaotriounssuaalsly tihenychmuasyednairo SS(ia"rpiosmboauctthirtoastm.attihde gaapm, l1o6%c)u.s iSnambeothasscihscreormatid Fcrauieluraeberorfaccihorno.matin packing. Probably not a Abianpdcleeolxlieddcno5unmtvbaeeirrnyin(gcno)mmmuooflnt.icphrloemocsoopmiees.s ofOntlhye P40iccsellhasinfawihliecdh. sebpulryatiionndexofedchLfromvoersyamecon. snes TM Chromaeid Break: SB Chromoscme Break: 4 E"rDaogunbelnet:Minutes ass ATnchaagnaacahcnrhtoemaavtaiiydcebhecoepfgair&ocnicshiinrloyomnroeirac.chormopaTnhlete.icdea,riyselegairiignieard io Ctnioheomranmogamnloneosnwo(mdeeithlnaechttlaesuddi)esasdccihtlsheeleoaomrcaoacsbteerenmedcsa.rk,iwciTifhhfoirrsaamgnimcneglsnectasanin(tca1iib)ce.acSTthherovamAsoFsonanossc aGpipfaarreennttlyfrreomtactehse s5o3 sonbptysihnactirroit odaTechrleeiesgtetiienoranmrsiaerneaaslnmdnaoldtleplrdeodicbosiautobbinllnsyegedasconmstadnsla,lsbomisseei.o,nmgeien.voferwaTchhiiesciihsal 16 of 18 ; 000904 Protocol No. 437, Edition 15 compLex I Incerscitial Deletion: TT teiradials QR quadriradial: CR Complex Rearrangement: D dicentric: or Tricenteic PC qPueandtraicceennttrriicc:: HC Hexacentric: Ro Ring: RC Ring Chromacid: re CT Chromosome Intrachange: T Translocation: ars CORNINGHazleton oOLffen&grcihncghrooflmyaictnhgirdombaertseiisdduelt"aicnugtshoionruttsaenesfdmraolcmlharoifmdrarategimgdeinotnor a gap in the chromatid cArnhersouemlxotcsshoamniegneaanbdtehccwaeeneen-aacretmnwetodriccchornoffmrioagsguomrmeaentsti,,onwo.hricohne Acsonftirgiurraadtiiaoln.. but resulting in a four-armed Anraegxncenhtasngewhiacmhongismotchee rtehsaunlttwoofcshervoemroaslomes or breaks. TAhneissuelxticsshanoigfneteanbceahtsrwsoeomeconisaottmweeodwcvihitrthohmotaswnoomeacsecnewtnrhtoeimicechres. ragnent in which case it ir classified as OF Dicentric with fragment mArenerseuse.lxtcihnagnOgfetinenianavsocslhovrcioinmagotseotdmherveiwetihtchhrtotomhoesteoomectsehnrtearenod-AF. aSnudchareexchnaanmgeedsascanfolilnovwoslve many chromosomes sfiioxvuerceccneetnnrttorrmooemmreeerrsee,ss., uupupptottoosfifioxvueArFAAFF Acencthreoommeorseo.ne Twhhiicshisfoormfstena acentric fragment in hich acisrscolceiatceodntwaiitnhingan& case it is classed as BF. Single chromatid ring (acentric). Ring with associated acentric fragment Etxhacthadnogeeswinotthinincalcudheromtohseomeen:tiree.gc.hroamocsionmge. hOSboevsnioomueissdenrtcerisafunilsstfbielnreg,ofinsmcaottrwoeerdiaabalsnorb"meTatlweneoctnhro<tm2voAosBo-c.mherso.- 17 of 10 000905 Protocol No. 437. Hition 13 COMPLEX (Continued) ome or CORNINGHazleton ACChhbeenoonrkamesacclyaoescrovpneho.ocseenatnmdroircopfhtcoehlnroogmtyohseoinefee.asubintoTrhmioasfl iasfora CaCslrcaarsnissbilefoditaccatitoroino,ne.upgea.ceic8eAmfrceracibicnporroimcnaavlleirestiyroancn.salmosecirca.tiboen. dGCxormneattgaeei:dns ctemhloalrne 1s0thohaualbder1z0baecaiabonenrasrl:aytsieosAnsvc.oellLAdwehhneicacithvyily NttahhveoeseS3y3pf0eo,sundeof.sgsa.sboemcruirlaattciieposlneswitafhnrdasgmmeatnyrtisncoetsnutacrhciucaastly ores 18 of 18 000906